This article was downloaded by: [University of Maastricht] On: 14 June 2014, At: 08:07 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Survey of ochratoxin A in rice from Iran using affinity column cleanup and HPLC with fluorescence detection a
a
a
J. Feizy , H.R. Beheshti , S.S. Fakoor Janati & N. Khoshbakht Fahim
a
a
Testa Quality Control Laboratory , North-East Food Industrial Technology and Biotechnology Park , Mashhad, Iran Published online: 24 Jan 2011.
To cite this article: J. Feizy , H.R. Beheshti , S.S. Fakoor Janati & N. Khoshbakht Fahim (2011) Survey of ochratoxin A in rice from Iran using affinity column cleanup and HPLC with fluorescence detection, Food Additives & Contaminants: Part B: Surveillance, 4:1, 67-70, DOI: 10.1080/19393210.2010.542252 To link to this article: http://dx.doi.org/10.1080/19393210.2010.542252
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives and Contaminants: Part B Vol. 4, No. 1, March 2011, 67–70
VIEW DATASET Survey of ochratoxin A in rice from Iran using affinity column cleanup and HPLC with fluorescence detection J. Feizy*, H.R. Beheshti, S.S. Fakoor Janati and N. Khoshbakht Fahim Testa Quality Control Laboratory, North-East Food Industrial Technology and Biotechnology Park, Mashhad, Iran (Received 17 October 2010; final version received 18 November 2010)
Downloaded by [University of Maastricht] at 08:07 14 June 2014
This paper records the occurrence and levels of ochratoxin A (OTA) in rice using a HPLC technique preceded by an immunoaffinity clean-up step. The method was based on the extraction of finely ground rice sample with an acetonitrile/water (60 : 40, v/v) solution. Recovery was 98.9% while the limit of detection (LOD) was 0.2 ng g1. A total of 182 rice samples were analyzed with a frequency of contamination of 6%. Levels of OTA in positive samples ranged 0.2–4.8 ng g–1, with an average contamination of all analyzed samples of 1.6 ng g–1. Keywords: rice; mycotoxins; ochratoxin A
Introduction Rice is the most consumed cereal in the world. The United Nations has launched a major international drive to increase production of rice, the staple food for more than half of the world’s population, and it declared 2004 the international year of rice (US Department of State, 2003). During cultivation and subsequent handling of rice, kernels can be contaminated by moulds, which can grow and produce mycotoxins if conditions are favorable. Fungal activity depends on moisture content and temperature, which can both vary significantly in a silo depending on its design and environmental factors. Post-harvest treatment of rice, including adequate drying and conditions of storage, are crucial factors determining storage stability (Fredlund et al. 2009). OTA is a mycotoxin produced by toxigenic mould species (Penicillium verrucosum, Aspergillus ochraceus, A. niger and A. carbonarius), which occurs in foods and animal feeds. OTA contaminates a large number of human foods, such as cereals, cacao, coffee, wine, fruits, peanuts, cotton seed, corn and rice, as a consequence of unfavourable storage conditions (humidity of 70–90% and a minimum temperature of 10 C) (Tarı´ n et al. 2004). OTA was classified as a possible human carcinogen (group 2B) by the International Agency for Research on Cancer from experimental studies demonstrating evidence for OTA carcinogenicity in animals (IARC 1993). The European Commission has enforced limits of OTA in cereals and cereal products with the following
*Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2011 Taylor & Francis DOI: 10.1080/19393210.2010.542252 http://www.informaworld.com
levels: 5 mg kg1 for raw cereal grains, 3 mg kg1 for cereals and cereal products intended for human consumption, 0.5 mg kg1 for baby food and cereal-based food intended for young children (European Commission 2006). There are currently no legal limits for OTA in spices; however, the European Commission has been discussing a limit of 10 mg kg1 for OTA in spices (Goryacheva et al. 2006). For dried vine fruits, soluble coffee and some dried fruits, the European Commission has set a maximum permissible limit for OTA of 10 mg kg1. Numerous methods for OTA determination in food have been described, including enzyme-linked immunosorbent assay (ELISA) and thin layer chromatography (TLC) (El-Kady et al. 1995). Liquid chromatography linked to fluorescence detection (HPLC/FD) is extensively used for OTA confirmatory analysis (Fazekas et al. 2005; Gonzalez et al. 2006). The natural occurrence of OTA has been reported from temperate, sub-tropical and tropical climates in several foods, including rice (Karin et al. 1998). Contamination of rice with OTA has been reported from the UK (Scudamore et al. 1999), Vietnam (Trung et al. 2001), Egypt (Abdelhamid 1990), Portugal (Pena et al. 2005), France (Leblanc et al. 2005), Korea (Park et al. 2005) and Spain (Gonzalez et al. 2006). Consumption of rice is widespread in Iran and, thus, this study was designed to determine the occurrence and levels of OTA, especially in rice sold and consumed in Khorasan Province.
68
J. Feizy et al.
Materials and methods Samples All samples were obtained locally from Mashhad (Khorasan, Iran). A minimum sample size of 1000 g was taken and samples were mixed for 10 min, then 500 g aliquots were ground to a fine powder and stored at 20 C while awaiting analysis. The particle sizes after grinding were 50.3 mm.
Downloaded by [University of Maastricht] at 08:07 14 June 2014
Reagents Methanol, acetonitrile, acetic acid and other chemical reagents were HPLC grade and supplied by Merck (Darmstadt, Germany). OTA was obtained from Sigma (St. Louis, MO, USA). Immunoaffinity columns (Neogen Europe, Scotland, UK) for the purification and pre-concentration of OTA prior to quantitative analysis were used. Double-distilled water was used in all the experiments. Stock standard solution of OTA with concentrations of 100 mg ml1 was prepared in methanol. Working standard solutions were prepared daily by diluting the stock solution with methanol/ acetic acid (98 : 2, v/v). All working standard solutions were stored in darkness in a refrigerator at 4 C.
Apparatus HPLC analyses were performed on a Sykam (Eresing, Germany) HPLC system equipped with an S2100 pump, an S7131 reagent organizer, an S4011 column thermo-controller, a RF-10Axl fluorescence detector and a ChromolithÕ performance RP18 analytical column (100 4.6 mm). The fluorescence detector was operated at 333 and 443 nm for excitation and emission, respectively. The guard column was ODS-H (20 4.6 mm). Clarity software was used for data management. For determination of OTA concentration, a UV–Visible spectrum of OTA stock solution against the solvent used as solution in the reference cell (Trucksess et al. 2008) was obtained using a Shimadzu UV-1700 Pharma (Tokyo, Japan) spectrophotometer equipped with a standard 10-mm path length cell.
Extraction Ground rice samples (25 g), either non-spiked or spiked with a known volume of an OTA stock solution, were mixed with 40 ml of pure water, 60 ml acetonitril and 0.3 g NaHCO3 and blended (Waring 8011S, Torrington, CT, USA) at high speed for 5 min to obtain a homogeneous sample mix. After mixing, the slurry was filtered though filter paper (Whatman No. 4) and a 10 ml aliquot mixed with 40 ml phosphate-buffered saline (PBS) solution. This diluted solution was filtered through a glass microfiber filter
(Whatman, Inc., Clifton, NJ, USA) and 55 ml was passed through the immunoaffinity column. OTA was eluted from the column by passing 1.5 ml of HPLCgrade methanol/acetic acid (98 : 2, v/v) and then 1.5 ml of HPLC-grade water and using gravity to collect the eluate into a glass vial at a flow-rate of 5 ml min1. Then, 20 ml of eluate was injected into the HPLC.
Chromatographic method Linear isocratic elution chromatography has done using water/acetonitrile/acetic acid (99 : 99 : 1, v/v) at 30 C as the solvent system. The flow rate was kept constant at 0.7 ml min1 and 20 ml was injected. OTA was detected by fluorescence detector (excitation 333 nm, emission 443 nm).
Calibration curve An external standard curve was constructed using reference standard OTA to quantify the OTA content in all samples. A stock working solution containing 100 ng ml1 OTA was prepared and then diluted to the appropriate concentration ranges with methanol/acetic acid (98 : 2, v/v). The calibration curve was constructed using five different concentrations with the square of the correlation coefficient (r2) equal to 0.999. These solutions covered the range 1–15 ng ml1. The calibration curve was derived by plotting concentrations as a function of peak area of OTA and curves exhibited good linear regression.
Results and discussion HPLC analysis Mycotoxin contamination is less commonly reported in rice than many other cereals. In this study, 182 rice samples were analyzed to evaluate the concentration of OTA. As shown in Table 1, OTA was detected in 8% of rice samples with a mean value 1.6 ng g1. All contaminated samples had a level of OTA below Iranian National Standard No. 5925 of 5 ng g1 (ISIRI 2002). Relevant data concerning the analytical system are summarized in Table 2. For OTA, LOD was 0.2 ng g1 and LOQ was 0.5 ng g1. For repeatability measurements, a standard solution containing 10 ng ml1 of OTA was used.
Recovery Accuracy was determined from the recoveries of OTA. The recovery study was performed by comparing the concentration in spiked samples to the respective nonextract standards (OTA in solution). The area under
Food Additives and Contaminants: Part B
69
Table 1. Analytical data for the OTA HPLC system.
Compound OTA
tR (min)
Repeatability (%RSD, n ¼ 7)
Limit of detection (ng g1)
Limit of quantification (ng g1)
Recovery
5.49
1.3
0.2
0.5
98.9%
Table 2. OTA in analyzed samples of rice.
Downloaded by [University of Maastricht] at 08:07 14 June 2014
OTA
Total
Positive samples
Average SD (ng g1)
Range (ng g1)
182
11
1.6 (1.6)
0.2–4.8
the peak of each sample was divided by the area under the curve of the quality control sample and multiplied by 100. Table 1 showed that the recovery of OTA from sample spiked at 5 ng g1. The recovery range was within the guidelines for acceptable recovery limits of the AOAC and Codex Alimentarius. The AOAC guideline for acceptable recovery at the 10 mg kg1 level is 70–125%. The Codex acceptable recovery range is 70–110% for a level of 10–100 mg kg1 and 60–120% for a level of 1–10 mg kg1 (Trucksess et al. 2008). Relative standard deviation for within-laboratory repeatability (RSDr, n ¼ 7) for all samples analyzed from beginning to end was 1.3%.
Conclusions Our study has shown that of 182 rice samples, 11 contained OTA with a mean value of 1.6 ng g1. The mean concentration of OTA contamination of rice from Mashhad was 1.6 ng g1, significantly below the accepted Iran regulation limit (5 ng g1). The present status of this mycotoxin in Iran is that it does not pose a serious risk to public health; nevertheless, there is a need for routine monitoring as a food quality control measure. Initial approaches to control the occurrence of OTA in rice have involved controlling contamination in the field; however, this is difficult as fungal growth is influenced primarily by climatic conditions, such as relative humidity and temperature.
Acknowledgements The authors thank the Testa Quality Control Laboratory (TQCL) for financial support.
References Abdelhamid AM. 1990. Occurrence of some mycotoxins (aflatoxins, ochratoxin A, citrinin, zearalenone and
vomitoxin) in various Egyptian feeds. Arch Anim Nutr. 40:647–664. El-Kady IA, El-Maraghy SS, Eman MM. 1995. Natural occurrence of mycotoxins in different spices in Egypt. Folia Microbiol. 40(3):297–300. European Commission. 2006. Commission Regulation, 1881/2006 of December 12th setting maximum levels of certain contaminants in foods. Off J Eur Commun. L 364: 5. Fazekas B, Tar A, Kovacs M. 2005. Aflatoxin and ochratoxin A content of spices in Hungary. Food Addit Contam. 22:856–863. Fredlund E, Thim AM, Gidlund A, Brostedt S, Nyberg M, Olsen M. 2009. Moulds and mycotoxins in rice from the Swedish retail market. Food Addit Contam. 26(4):527–533. Gonzalez L, Juan C, Soriano JM, Molto JC, Manes J. 2006. Occurrence and daily intake of ochratoxin A of organic and non-organic rice and rice products. Int J Food Microbiol. 107:223–227. Goryacheva IY, De Saeger S, Lobeau M, Eremin SA, BarnaVetro I, Van PC. 2006. Approach for ochratoxin A fast screening in spices using clean-up tandem immunoassay columns with confirmation by high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/ MS). Anal Chim Acta. 577:38–45. IARC. 1993. Evaluation of carcinogenic risks of chemical to humans. IARC monograph on some naturally-occurring substances: Food Items and Constituents’’. Heterocyclic Aromatic Amines and Mycotoxins. Lyon, France: IARC. p. 359–362. ISIRI. 2002. Food and feed. Mycotoxins: Maximum tolerated level. 1st ed. Institute of Standards and Industrial Research of Iran publication No. 5925. Karin A, Gunnar J, Karl H. 1998. Ochratoxin A in rice cultivars after inoculation of Penicillium verrucosum. Nat Toxins. 6:73–84. Leblanc JC, Tard A, Volatier JL, Verger P. 2005. Estimated dietary exposure to principal food mycotoxins from The First French Total Diet Study. Food Addit Contam. 22:652–672. Park JW, Choi SY, Hwang HJ, Kim YB. 2005. Fungal mycoflora and mycotoxins in Korean polished rice destined for humans. Int J Food Microbiol. 103:305–314. Pena A, Cerejo F, Lino C, Silveira I. 2005. Determination of ochratoxin A in Portuguese rice samples by high performance liquid chromatography with fluorescence detection. Anal Bioanal Chem. 382:1288–1293. Scudamore KA, Patel S, Breeze V. 1999. Surveillance of stored grain from the 1997 harvest in the United Kingdom for ochratoxin A. Food Addit Contam. 16:281–290. Tarı´ n A, Rosell MG, Guardino X. 2004. Use of highperformance liquid chromatography to assess airborne
70
J. Feizy et al.
Downloaded by [University of Maastricht] at 08:07 14 June 2014
mycotoxins Aflatoxins and ochratoxin A. J Chromatogr A. 1047:235–240. Trucksess MW, Weaver CM, Oles CJ, Fry FS Jr, Noonan GO, Betz JM, Reader JI. 2008. Determination of aflatoxins B1, B2, G1, and G2 and ochratoxin A in ginseng and ginger by multitoxin immunoaffinity column cleanup and liquid chromatographic quantitation: collaborative study. J AOAC Int. 91(3):511–523.
Trung TS, Bailly JD, Querin A, Lebars P, Guerre P. 2001. Fungal contamination of rice from South Vietnam, mycotoxigenesis of selected strains and residues in rice. Rev Me´d. Ve´t. 152:555–560. US Department of State. 2003. UN declares 2004 International Year of Rice. Washington (DC): US Department of State. Available from: http://usinfo.state. gov/gi/Archive/2003/Nov/05-674995.html
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Survey of ochratoxin A in rice from Iran using affinity column cleanup and HPLC with fluorescence detection a
a
a
J. Feizy , H.R. Beheshti , S.S. Fakoor Janati & N. Khoshbakht Fahim
a
a
Testa Quality Control Laboratory , North-East Food Industrial Technology and Biotechnology Park , Mashhad, Iran Published online: 24 Jan 2011.
To cite this article: J. Feizy , H.R. Beheshti , S.S. Fakoor Janati & N. Khoshbakht Fahim (2011) Survey of ochratoxin A in rice from Iran using affinity column cleanup and HPLC with fluorescence detection, Food Additives & Contaminants: Part B: Surveillance, 4:1, 67-70, DOI: 10.1080/19393210.2010.542252 To link to this article: http://dx.doi.org/10.1080/19393210.2010.542252
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives and Contaminants: Part B Vol. 4, No. 1, March 2011, 67–70
VIEW DATASET Survey of ochratoxin A in rice from Iran using affinity column cleanup and HPLC with fluorescence detection J. Feizy*, H.R. Beheshti, S.S. Fakoor Janati and N. Khoshbakht Fahim Testa Quality Control Laboratory, North-East Food Industrial Technology and Biotechnology Park, Mashhad, Iran (Received 17 October 2010; final version received 18 November 2010)
Downloaded by [University of Maastricht] at 08:07 14 June 2014
This paper records the occurrence and levels of ochratoxin A (OTA) in rice using a HPLC technique preceded by an immunoaffinity clean-up step. The method was based on the extraction of finely ground rice sample with an acetonitrile/water (60 : 40, v/v) solution. Recovery was 98.9% while the limit of detection (LOD) was 0.2 ng g1. A total of 182 rice samples were analyzed with a frequency of contamination of 6%. Levels of OTA in positive samples ranged 0.2–4.8 ng g–1, with an average contamination of all analyzed samples of 1.6 ng g–1. Keywords: rice; mycotoxins; ochratoxin A
Introduction Rice is the most consumed cereal in the world. The United Nations has launched a major international drive to increase production of rice, the staple food for more than half of the world’s population, and it declared 2004 the international year of rice (US Department of State, 2003). During cultivation and subsequent handling of rice, kernels can be contaminated by moulds, which can grow and produce mycotoxins if conditions are favorable. Fungal activity depends on moisture content and temperature, which can both vary significantly in a silo depending on its design and environmental factors. Post-harvest treatment of rice, including adequate drying and conditions of storage, are crucial factors determining storage stability (Fredlund et al. 2009). OTA is a mycotoxin produced by toxigenic mould species (Penicillium verrucosum, Aspergillus ochraceus, A. niger and A. carbonarius), which occurs in foods and animal feeds. OTA contaminates a large number of human foods, such as cereals, cacao, coffee, wine, fruits, peanuts, cotton seed, corn and rice, as a consequence of unfavourable storage conditions (humidity of 70–90% and a minimum temperature of 10 C) (Tarı´ n et al. 2004). OTA was classified as a possible human carcinogen (group 2B) by the International Agency for Research on Cancer from experimental studies demonstrating evidence for OTA carcinogenicity in animals (IARC 1993). The European Commission has enforced limits of OTA in cereals and cereal products with the following
*Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2011 Taylor & Francis DOI: 10.1080/19393210.2010.542252 http://www.informaworld.com
levels: 5 mg kg1 for raw cereal grains, 3 mg kg1 for cereals and cereal products intended for human consumption, 0.5 mg kg1 for baby food and cereal-based food intended for young children (European Commission 2006). There are currently no legal limits for OTA in spices; however, the European Commission has been discussing a limit of 10 mg kg1 for OTA in spices (Goryacheva et al. 2006). For dried vine fruits, soluble coffee and some dried fruits, the European Commission has set a maximum permissible limit for OTA of 10 mg kg1. Numerous methods for OTA determination in food have been described, including enzyme-linked immunosorbent assay (ELISA) and thin layer chromatography (TLC) (El-Kady et al. 1995). Liquid chromatography linked to fluorescence detection (HPLC/FD) is extensively used for OTA confirmatory analysis (Fazekas et al. 2005; Gonzalez et al. 2006). The natural occurrence of OTA has been reported from temperate, sub-tropical and tropical climates in several foods, including rice (Karin et al. 1998). Contamination of rice with OTA has been reported from the UK (Scudamore et al. 1999), Vietnam (Trung et al. 2001), Egypt (Abdelhamid 1990), Portugal (Pena et al. 2005), France (Leblanc et al. 2005), Korea (Park et al. 2005) and Spain (Gonzalez et al. 2006). Consumption of rice is widespread in Iran and, thus, this study was designed to determine the occurrence and levels of OTA, especially in rice sold and consumed in Khorasan Province.
68
J. Feizy et al.
Materials and methods Samples All samples were obtained locally from Mashhad (Khorasan, Iran). A minimum sample size of 1000 g was taken and samples were mixed for 10 min, then 500 g aliquots were ground to a fine powder and stored at 20 C while awaiting analysis. The particle sizes after grinding were 50.3 mm.
Downloaded by [University of Maastricht] at 08:07 14 June 2014
Reagents Methanol, acetonitrile, acetic acid and other chemical reagents were HPLC grade and supplied by Merck (Darmstadt, Germany). OTA was obtained from Sigma (St. Louis, MO, USA). Immunoaffinity columns (Neogen Europe, Scotland, UK) for the purification and pre-concentration of OTA prior to quantitative analysis were used. Double-distilled water was used in all the experiments. Stock standard solution of OTA with concentrations of 100 mg ml1 was prepared in methanol. Working standard solutions were prepared daily by diluting the stock solution with methanol/ acetic acid (98 : 2, v/v). All working standard solutions were stored in darkness in a refrigerator at 4 C.
Apparatus HPLC analyses were performed on a Sykam (Eresing, Germany) HPLC system equipped with an S2100 pump, an S7131 reagent organizer, an S4011 column thermo-controller, a RF-10Axl fluorescence detector and a ChromolithÕ performance RP18 analytical column (100 4.6 mm). The fluorescence detector was operated at 333 and 443 nm for excitation and emission, respectively. The guard column was ODS-H (20 4.6 mm). Clarity software was used for data management. For determination of OTA concentration, a UV–Visible spectrum of OTA stock solution against the solvent used as solution in the reference cell (Trucksess et al. 2008) was obtained using a Shimadzu UV-1700 Pharma (Tokyo, Japan) spectrophotometer equipped with a standard 10-mm path length cell.
Extraction Ground rice samples (25 g), either non-spiked or spiked with a known volume of an OTA stock solution, were mixed with 40 ml of pure water, 60 ml acetonitril and 0.3 g NaHCO3 and blended (Waring 8011S, Torrington, CT, USA) at high speed for 5 min to obtain a homogeneous sample mix. After mixing, the slurry was filtered though filter paper (Whatman No. 4) and a 10 ml aliquot mixed with 40 ml phosphate-buffered saline (PBS) solution. This diluted solution was filtered through a glass microfiber filter
(Whatman, Inc., Clifton, NJ, USA) and 55 ml was passed through the immunoaffinity column. OTA was eluted from the column by passing 1.5 ml of HPLCgrade methanol/acetic acid (98 : 2, v/v) and then 1.5 ml of HPLC-grade water and using gravity to collect the eluate into a glass vial at a flow-rate of 5 ml min1. Then, 20 ml of eluate was injected into the HPLC.
Chromatographic method Linear isocratic elution chromatography has done using water/acetonitrile/acetic acid (99 : 99 : 1, v/v) at 30 C as the solvent system. The flow rate was kept constant at 0.7 ml min1 and 20 ml was injected. OTA was detected by fluorescence detector (excitation 333 nm, emission 443 nm).
Calibration curve An external standard curve was constructed using reference standard OTA to quantify the OTA content in all samples. A stock working solution containing 100 ng ml1 OTA was prepared and then diluted to the appropriate concentration ranges with methanol/acetic acid (98 : 2, v/v). The calibration curve was constructed using five different concentrations with the square of the correlation coefficient (r2) equal to 0.999. These solutions covered the range 1–15 ng ml1. The calibration curve was derived by plotting concentrations as a function of peak area of OTA and curves exhibited good linear regression.
Results and discussion HPLC analysis Mycotoxin contamination is less commonly reported in rice than many other cereals. In this study, 182 rice samples were analyzed to evaluate the concentration of OTA. As shown in Table 1, OTA was detected in 8% of rice samples with a mean value 1.6 ng g1. All contaminated samples had a level of OTA below Iranian National Standard No. 5925 of 5 ng g1 (ISIRI 2002). Relevant data concerning the analytical system are summarized in Table 2. For OTA, LOD was 0.2 ng g1 and LOQ was 0.5 ng g1. For repeatability measurements, a standard solution containing 10 ng ml1 of OTA was used.
Recovery Accuracy was determined from the recoveries of OTA. The recovery study was performed by comparing the concentration in spiked samples to the respective nonextract standards (OTA in solution). The area under
Food Additives and Contaminants: Part B
69
Table 1. Analytical data for the OTA HPLC system.
Compound OTA
tR (min)
Repeatability (%RSD, n ¼ 7)
Limit of detection (ng g1)
Limit of quantification (ng g1)
Recovery
5.49
1.3
0.2
0.5
98.9%
Table 2. OTA in analyzed samples of rice.
Downloaded by [University of Maastricht] at 08:07 14 June 2014
OTA
Total
Positive samples
Average SD (ng g1)
Range (ng g1)
182
11
1.6 (1.6)
0.2–4.8
the peak of each sample was divided by the area under the curve of the quality control sample and multiplied by 100. Table 1 showed that the recovery of OTA from sample spiked at 5 ng g1. The recovery range was within the guidelines for acceptable recovery limits of the AOAC and Codex Alimentarius. The AOAC guideline for acceptable recovery at the 10 mg kg1 level is 70–125%. The Codex acceptable recovery range is 70–110% for a level of 10–100 mg kg1 and 60–120% for a level of 1–10 mg kg1 (Trucksess et al. 2008). Relative standard deviation for within-laboratory repeatability (RSDr, n ¼ 7) for all samples analyzed from beginning to end was 1.3%.
Conclusions Our study has shown that of 182 rice samples, 11 contained OTA with a mean value of 1.6 ng g1. The mean concentration of OTA contamination of rice from Mashhad was 1.6 ng g1, significantly below the accepted Iran regulation limit (5 ng g1). The present status of this mycotoxin in Iran is that it does not pose a serious risk to public health; nevertheless, there is a need for routine monitoring as a food quality control measure. Initial approaches to control the occurrence of OTA in rice have involved controlling contamination in the field; however, this is difficult as fungal growth is influenced primarily by climatic conditions, such as relative humidity and temperature.
Acknowledgements The authors thank the Testa Quality Control Laboratory (TQCL) for financial support.
References Abdelhamid AM. 1990. Occurrence of some mycotoxins (aflatoxins, ochratoxin A, citrinin, zearalenone and
vomitoxin) in various Egyptian feeds. Arch Anim Nutr. 40:647–664. El-Kady IA, El-Maraghy SS, Eman MM. 1995. Natural occurrence of mycotoxins in different spices in Egypt. Folia Microbiol. 40(3):297–300. European Commission. 2006. Commission Regulation, 1881/2006 of December 12th setting maximum levels of certain contaminants in foods. Off J Eur Commun. L 364: 5. Fazekas B, Tar A, Kovacs M. 2005. Aflatoxin and ochratoxin A content of spices in Hungary. Food Addit Contam. 22:856–863. Fredlund E, Thim AM, Gidlund A, Brostedt S, Nyberg M, Olsen M. 2009. Moulds and mycotoxins in rice from the Swedish retail market. Food Addit Contam. 26(4):527–533. Gonzalez L, Juan C, Soriano JM, Molto JC, Manes J. 2006. Occurrence and daily intake of ochratoxin A of organic and non-organic rice and rice products. Int J Food Microbiol. 107:223–227. Goryacheva IY, De Saeger S, Lobeau M, Eremin SA, BarnaVetro I, Van PC. 2006. Approach for ochratoxin A fast screening in spices using clean-up tandem immunoassay columns with confirmation by high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/ MS). Anal Chim Acta. 577:38–45. IARC. 1993. Evaluation of carcinogenic risks of chemical to humans. IARC monograph on some naturally-occurring substances: Food Items and Constituents’’. Heterocyclic Aromatic Amines and Mycotoxins. Lyon, France: IARC. p. 359–362. ISIRI. 2002. Food and feed. Mycotoxins: Maximum tolerated level. 1st ed. Institute of Standards and Industrial Research of Iran publication No. 5925. Karin A, Gunnar J, Karl H. 1998. Ochratoxin A in rice cultivars after inoculation of Penicillium verrucosum. Nat Toxins. 6:73–84. Leblanc JC, Tard A, Volatier JL, Verger P. 2005. Estimated dietary exposure to principal food mycotoxins from The First French Total Diet Study. Food Addit Contam. 22:652–672. Park JW, Choi SY, Hwang HJ, Kim YB. 2005. Fungal mycoflora and mycotoxins in Korean polished rice destined for humans. Int J Food Microbiol. 103:305–314. Pena A, Cerejo F, Lino C, Silveira I. 2005. Determination of ochratoxin A in Portuguese rice samples by high performance liquid chromatography with fluorescence detection. Anal Bioanal Chem. 382:1288–1293. Scudamore KA, Patel S, Breeze V. 1999. Surveillance of stored grain from the 1997 harvest in the United Kingdom for ochratoxin A. Food Addit Contam. 16:281–290. Tarı´ n A, Rosell MG, Guardino X. 2004. Use of highperformance liquid chromatography to assess airborne
70
J. Feizy et al.
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